Microencapsulation and sustained release of biologically active polypeptides

ABSTRACT

This invention relates to compositions for the sustained release of biologically active polypeptides, and methods of forming and using said compositions, for the sustained release of biologically active polypeptides. The sustained release compositions of this invention comprise a biocompatible polymer having dispersed therein, a biologically active polypeptide, a sugar and a salting-out salt.

RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/419,388, filed Oct. 17, 2002.

[0002] The entire teachings of the above application(s) are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0003] Numerous proteins and peptides, collectively referred to hereinas polypeptides, exhibit biological activity in vivo, which renders themuseful as medicaments in therapy. Many illnesses or conditions which canbe treated with a biologically active polypeptide, requireadministration of a constant or sustained level of medicament to providethe most effective prophylactic and/or therapeutic effects. As such,administration of biologically active polypeptides can require frequentsubcutaneous injections, which result in fluctuating levels ofmedicament and poor patient compliance.

[0004] As an alternative, the use of biodegradable materials, such aspolymers, encapsulating the medicament can be employed as a sustaineddelivery system. The use of biodegradable polymers, for example, in theform of microparticles or microcarriers, can provide a sustained releaseof medicament, by utilizing the inherent biodegradability of the polymerto control the release of the medicament thereby providing a moreconsistent, sustained level of medicament and improved patientcompliance.

[0005] However, these sustained release devices can exhibit high initialbursts of medicament and minimal release thereafter, resulting in serumdrug levels outside the therapeutic window and/or poor bioavailabilityof the medicament. In addition, the presence of polymer, physiologicaltemperatures and body response to the sustained release composition cancause the medicament to be altered (e.g., degraded, aggregated) therebyinterfering with the desired release profile for the medicament.

[0006] Further, methods used to form sustained release compositions canresult in loss of activity of the medicament due to the instability ofthe medicament and the degradative effects of the processing steps.Degradative effects are particularly problematic when the medicament isa polypeptide.

[0007] Therefore, a need exists for a means of administeringbiologically active polypeptides in a sustained fashion wherein theamount of polypeptide delivered is at therapeutic levels, and retainsactivity and potency for the desired period of release.

SUMMARY OF THE INVENTION

[0008] The present invention is based upon the unexpected discovery thatwhen a specific combination of excipients, namely a sugar andsalting-out salt, are present in a sustained release compositioncomprising a biocompatible polymer and a biologically active polypeptidethat an improved release profile of active agent is achieved. Mostnotably, sustained release compositions having this specific combinationof excipients exhibit an increase of bioavailability of the polypeptideover compositions lacking the specific combination of excipients,thereby resulting in an improved sustained release composition which candeliver therapeutic levels of polypeptide for a desired period. Inaddition, the sustained release compositions having this specificcombination of excipients can exhibit a reduced lag phase which canprovide for a smoothing out of the release profile and can contribute toan increase in the amount of agent released.

[0009] This invention relates to compositions for the sustained releaseof biologically active polypeptides, and methods of forming and usingsaid compositions for the sustained release of biologically activepolypeptides. The sustained release compositions of this inventioncomprise a biocompatible polymer having dispersed therein, abiologically active polypeptide, a sugar and a salting-out salt.

[0010] The method of the invention, for forming a composition for thesustained release of biologically active polypeptide, includesdissolving a biocompatible polymer in a polymer solvent to form apolymer solution, and combining a biologically active polypeptide, sugarand salting-out salt with the polymer solution. The biologically activepolypeptide, sugar and salting-out salt can be combined with the polymersolution either alone or in a premixed form. The biologically activepolypeptide, sugar and salting-out salt can be combined with the polymersolution either as solids, liquids or suspensions. It is understood thatthe method of combining the polymer, active and excipients can beperformed in any order.

[0011] The method of using the sustained release composition of thepresent invention comprises providing a therapeutically effective bloodlevel of biologically active polypeptide, in a patient for a sustainedperiod by administering to the patient a dose of the sustained releasecomposition described herein.

[0012] In a particular embodiment, the sustained release composition hasincorporated therein a hormone, particularly an anti-diabetic orglucoregulatory peptide, for example, GLP-1, GLP-2, exendin-3, exendin-4or agonists, analogs or derivatives thereof and the composition isadministered in a therapeutically effective amount to treat a patientsuffering from diabetes mellitus, impaired glucose tolerance (IGT),obesity, cardiovascular (CV) disorder or any other disorder that can betreated by one of the above polypeptides or derivatives, analogs oragonists thereof.

[0013] The sustained release composition of the invention overcomes theproblem of lack of acceptable bioavailability of the incorporatedbiologically active polypeptide which can occur when the specificcombination of sugar and salting-out salt is not present in thesustained release composition. Further, loss of activity of thebiologically active polypeptide due to instability and/or chemicalinteractions between the biologically active polypeptide and othercomponents which are contained in or used in formulating the sustainedrelease composition, can be minimized.

[0014] The advantages of the sustained release formulation forbiologically active molecule as described herein include increasedpatient compliance and acceptance by eliminating the need for repetitiveadministration, increased therapeutic benefit by eliminatingfluctuations in active agent concentration in blood levels by providinga desirable release profile, and a potential lowering of the totalamount of biologically active polypeptide necessary to provide atherapeutic benefit by reducing these fluctuations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0016]FIG. 2A is a graph of % Native Exendin-4 versus time in days forthe described stability studies.

[0017]FIG. 2B is a graph of % Native Exendin-4 versus time in days forthe described stability studies.

[0018]FIG. 3 is a graph of % Native Exendin-4 versus time in days forthe described stability studies.

[0019]FIG. 4 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0020]FIG. 5 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0021]FIG. 6 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0022]FIG. 7 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0023]FIG. 8 is a graph of plasma levels (pg/mL) for formulations, IF-1,-2 and -4 in Rats versus time in days post administration.

[0024]FIG. 9 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for formulations SF-1 and SF-2.

[0025]FIG. 10 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0026]FIG. 11 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0027]FIG. 12 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0028]FIG. 13 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0029]FIG. 14 is a graph of plasma levels (pg/1 nL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0030]FIG. 15 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0031]FIG. 16 is a graph of plasma levels (pg/mL) in rats of exendin-4versus time in days post administration for the selected microparticleformulations.

[0032]FIG. 17 is a graph of serum exendin-4 levels (pg/mL) in ratsadministered 120 mg of exendin-containing microparticles plus 30 mg ofplacebo microparticles or 10 mg of 2% triamcinolone acetonide-containingmicroparticles versus time in days.

[0033]FIG. 18 is a graph of serum exendin-4 levels (pg/mL) in ratsadministered 40 mg of exendin-containing microparticles plus 30 mg ofplacebo microparticles or 10 mg of 2% triamcinolone acetonide-containingmicroparticles versus time in days.

DETAILED DESCRIPTION OF THE INVENTION

[0034] This invention relates to compositions for the sustained releaseof biologically active polypeptides, and methods of forming and usingsaid compositions, for the sustained release of biologically activepolypeptides. The sustained release compositions of this inventioncomprise a biocompatible polymer having dispersed therein, abiologically active polypeptide, a sugar and a salting-out salt.

[0035] The method of the invention, for forming a composition for thesustained release of biologically active polypeptide, includesdissolving a biocompatible polymer in a polymer solvent to form apolymer solution, and combining a biologically active polypeptide aloneor in combination with other active ingredients, sugar and salting-outsalt with the polymer solution. The biologically active polypeptide,sugar and salting-out salt can be combined with the polymer solutioneither alone or in a premixed form. The biologically active polypeptide,sugar and salting-out salt can be combined with the polymer solutioneither as solids, liquids or suspensions. It is understood, that thecombination of the components of the composition can be achieved in anyorder.

[0036] The method of using the sustained release composition of thepresent invention comprises providing a therapeutically effective bloodlevel of biologically active polypeptide, in a patient for a sustainedperiod by administering to the patient a dose of the sustained releasecomposition described herein.

[0037] In a particular embodiment, the sustained release compositioncomprises a biocompatible polymer, an antidiabetic or glucoregulatorypolypeptide, a sugar and a salting-out salt. More specifically, thepolypeptide is selected from GLP-1, GLP-2, exendin-3, exendin-4 or ananalog, derivative or agonist thereof. Most specifically, thepolypeptide is exendin-4. When the polypeptide is exendin-4, the sugaris preferably sucrose, mannitol or a combination thereof and thesalting-out salt is preferably ammonium sulfate. This preferredcombination in no way excludes other combinations of sugar or salt withexendin-4 and it is understood that other combinations are included.

[0038] In a particular embodiment, the sustained release compositioncomprises a biocompatible polymer, exendin-4, sucrose and a salting-outsalt. The exendin-4 can be present in the composition at a concentrationof about 0.01% to about 10% w/w based on the total weight of the finalcomposition. In addition, the sucrose can be present in a concentrationof about 0.01% to about 5% w/w of the dry weight of the composition.Further, the ammonium sulfate can be present in the sustained releasecomposition at a concentration of about 0.01% to about 5% w/w of thefinal weight of the composition.

[0039] Salting-out salts, as that term is used herein, refers to saltswhich are in the Hofineister series of precipitants of serum globulins(or “salting-out salts”) as described in Thomas E. Creighton inProteins: Structures and Molecular Principles, pp. 149-150 (published byW.H. Freeman and Company, New York). In general, the salting-out saltsare known in the art as suitable for precipitating a protein, withoutdenaturing the protein. Salting-out salts can also be described in termsof the “kosmotrope” and “chaotrope” properties of the constituent ions.The term kosmotrope generally refers to a solute that stabilizesproteins and chaotrope describes a solute that is destabilizing.Kosmotropic ions have a high charge density (e.g., SO₄ ², HPO₄ ², Mg²⁺,Ca²⁺, Li⁺, Na⁺ and HPO₄ ²⁻) and chaotropic ions have a low chargedensity (examples include H₂PO₄ ⁻, HSO₄ ²⁻, HCO₃ ⁻, I⁻, Cl⁻, NO₃ ⁻, NH₄⁺, Cs⁺, K⁺, [N(CH₃)₄]⁺). The salting out salt can also be described interms of its ability to donate or accept protons, and as such acting asa base or acid. For instance, the salting out salt (NH₄)₂SO₄ provides anammonium ion, and can act as an inorganic acid. When included in apolymeric microparticle such inorganic acids can modulate polymerdegradation and effect release of incorporated agent. In certainembodiments, amino acids such as glycine which is considered in the artas a kosmotrope can be used as an alternative to the salting-out salt.

[0040] Suitable salting-out salts for use in this invention include, forexample, salts containing one or more of the cations Mg⁺², Li⁺, Na⁺, K⁺and NH₄ ⁺; and also containing one or more of the anions SO₄ ⁻², HPO₄⁻², acetate, citrate, tartrate, Cl⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻ and SCN⁻.

[0041] The amount of salting-out salt present in the sustained releasecomposition can range from about 0.01% (w/w) to about 50% (w/w), such asfrom about 0.01% to about 10% (w/w), for example from about 0.01% toabout 5%, such as 0.1% to about 5% of the total weight of the sustainedrelease composition. Combinations of two or more salting-out salts canbe used. The amount of salting-out salt, when a combination is employed,is the same as the range recited above.

[0042] A sugar as defined herein, is a monosaccharide, disaccharide oroligosaccharide (from 3-10 monosaccharides) or a derivative thereof. Forexample, sugar alcohols of monosaccharides are suitable derivativesincluded in the present definition of sugar. As such, the sugar alcoholmannitol, for example, which is derived from the monosaccharide mannoseis included in the definition of sugar as used herein.

[0043] Suitable monosaccharides include, but are not limited to,glucose, fructose and mannose. A disaccharide, as further definedherein, is a compound which upon hydrolysis yields two molecules of amonosaccharide. Suitable disaccharides include, but are not limited to,sucrose, lactose and trehalose. Suitable oligosaccharides include, butare not limited to, raffinose and acarbose.

[0044] The amount of sugar present in the sustained release compositioncan range from about 0.01% (w/w) to about 50% (w/w), such as from about0.01% (w/w) to about 10% (w/w), such as from about 0.1% (w/w) to about5% (w/w) of the total weight of the sustained release composition.

[0045] Alternatively, the amount of sugar present in the sustainedrelease composition can be referred to on a weight ratio with thebiologically active polypeptide. For example, the polypeptide and sugarcan be present in a ratio from about 10:1 to about 1:10 weight:weight.

[0046] Combinations of two or more sugars can also be used. The amountof sugar, when a combination is employed, is the same as the rangesrecited above.

[0047] Biologically active polypeptides as used herein collectivelyrefers to biologically active proteins and peptides and thepharmaceutically acceptable salts thereof, which are in their molecular,biologically active form when released in vivo, thereby possessing thedesired therapeutic, prophylactic and/or diagnostic properties in vivo.Typically, the polypeptide has a molecular weight between 500 and200,000 Daltons.

[0048] Suitable biologically active polypeptides include, but are notlimited to, glucagon, glucagon-like peptides such as, GLP-1, GLP-2,exendin-3, exendin-4 and analogs, agonists, and derivatives thereof,vasoactive intestinal peptide (VIP), immunoglobulins, antibodies,cytokines (e.g., lymphokines, monokines, chemokines), interleukins,macrophage activating factors, interferons, erythropoietin, nucleases,tumor necrosis factor, colony stimulating factors (e.g., G-CSF),insulin, enzymes (e.g., superoxide dismutase, plasminogen activator,etc.), tumor suppressors, blood proteins, hormones and hormone analogsand agonists (e.g., follicle stimulating hormone, growth hormone,adrenocorticotropic hormone, and luteinizing hormone releasing hormone(LHRH)), vaccines (e.g., tumoral, bacterial and viral antigens),antigens, blood coagulation factors, growth factors (NGF and EGF),gastrin, GRH, antibacterial peptides such as defensin, enkephalins,bradykinins, calcitonin and muteins, analogs, deletion and substitutionvariants and pharmaceutically acceptable salts of the foregoing.

[0049] Exendin-4 is a 39 amino acid polypeptide. The amino acid sequenceof exendin-4 can be found in U.S. Pat. No. 5,424,286 issued to Eng onJun. 13, 1995, the entire content of which is hereby incorporated byreference. AC2993 is synonymous with the term exendin-4 simply referringto the synthetic exendin-4. Exendin-4 has been shown in humans andanimals to stimulate secretion of insulin in the presence of elevatedblood glucose concentrations, but not during periods of low bloodglucose concentrations (hypoglycemia). It has also been shown tosuppress glucagon secretion, slow gastric emptying and affect foodintake and satiety, as well as other actions. As such, exendin-4 andanalogs and agonists thereof can be useful in the treatment of diabetesmellitus, IGT, obesity, etc.

[0050] The amount of biologically active polypeptide, which is containedwithin the polymeric matrix of a sustained release composition, is atherapeutically, diagnostically or prophylactically effective amountwhich can be determined by a person of ordinary skill in the art, takinginto consideration factors such as body weight, condition to be treated,type of polymer used, and release rate from the polymer.

[0051] Typically, the sustained release composition can contain fromabout 0.01% (w/w) to about 50% (w/w) of the biologically activepolypeptide (total weight of composition). For example, the amount ofbiologically active polypeptide can be from about 0.1%(w/w) to about 30%(w/w) of the total weight of the composition. The amount of polypeptidewill vary depending upon the desired effect, the planned release levels,and the time span over which the polypeptide will be released. Aspecific range of loading is between about 0.1% (w/w) to about 10%(w/w), for example, 0.5% (w/w) to about 5% (w/w).

[0052] In a further embodiment, the sustained release composition of thepresent invention can be coadministered with a corticosteroid. It hasbeen shown herein that coadministration of the sustained releasecomposition of the invention with a corticosteroid can further increasethe bioavailablity of the biologically active polypeptide of thesustained release composition. Coadministration of a corticosteroid incombination with sustained release compositions is described in detailin U.S. patent application having U.S. Ser. No. 60/419,430 entitled,Method of Modifying the Release Profile of Sustained ReleaseCompositions by Dasch et al. being filed concurrently, the entirecontent of which is hereby incorporated by reference.

[0053] Corticosteroids, as defined herein, refers to steroidalanti-inflammatory agents also referred to as glucocorticoids.

[0054] Suitable corticosteroids include, but are not limited to,21-Acetoxypregnenolone, Alclometasone, Algestone, Amcinonide,Beclomethasone, Betamethasone, Budesonide, Chloroprednisone, Clobetasol,Clobetasone, Clocortolone, Cloprednol, Corticosterone, Cortisone,Cortivazol, Deflazacort, Desonide, Desoximetasone, Dexamethasone,Disflorasone, Diflucortolone, Difluprednate, Enoxolone, Fluazacort,Flucloronide, Flumethasone, Flunisolide, Flucinolone Acetonide,Fluocinonide, Fluocortin Butyl, Flucortolone, Fluorometholone,Fluperolone Acetate, Fluprednidene Acetate, Fluprednisolone,Flurandrenolide, Fluticasone Propionate, Formocortal, Halcinonide,Halobetasol Propionate, Halometasone, Halopredone Acetate,Hydrocortamate, Hydrocortisone, Loteprednol Etabonate, Mazipredone,Medrysone, Meprednisone, Methylprednisolone, Mometasone Furoate,Paramethasone, Prednicarbate, Prednisolone, Prednisolone25-Diethylamino-acetate, Prednisolone Sodium Phosphate, Prednisone,Prednival, Prednylidene, Rimexolone, Tixocortol, Triamcinolone (allforms), for example, Triamcinolone Acetonide, Triamcinolone Acetonide21-oic acid methyl ester, Triamcinolone Benetonide, TriamcinoloneHexacetonide, Triamcinolone Diacetate, pharmaceutically acceptablemixtures thereof and salts thereof and any other derivative and analogthereof.

[0055] In one embodiment, the corticosteroid can be co-incorporated intothe sustained release composition comprising the biocompatible polymerand the biologically active polypeptide agent incorporated therein.

[0056] In another embodiment, the corticosteroid can be separatelyincorporated into a second biocompatible polymer. The secondbiocompatible polymer can be the same or different from the firstbiocompatible polymer which has the biologically active polypeptideagent incorporated therein.

[0057] In yet another embodiment, the corticosteroid can be present inan unencapsulated state but commingled with the sustained releasecomposition. For example, the corticosteroid can be solubilized in thevehicle used to deliver the sustained release composition.Alternatively, the corticosteroid can be present as a solid suspended inan appropriate vehicle. Further, the corticosteroid can be present as apowder which is commingled with the sustained release composition.

[0058] It is understood that the corticosteroid is present in an amountsufficient to modify the release profile of the biologically activepolypeptide from the sustained release composition. Modification of therelease profile, refers to increased bioavailability of the biologicallyactive polypeptide of the sustained release composition. Increasedbioavailability refers to an increase in the bioavailability of thebiologically active polypeptide from the sustained release compositionwhen coadministered with a corticosteroid in comparison to theadministration in the absence of corticosteroid over a time periodbeginning at two days post administration and ending at the targetedtimepoint for the particular formulation.

[0059] As used herein, the term a or an refer to one or more.

[0060] As used herein, patient refers to a human.

[0061] As defined herein, a sustained release of biologically activepolypeptide is a release of the polypeptide from the sustained releasecomposition of the invention which occurs over a period which is longerthan that period during which a biologically significant amount of thepolypeptide would be available following direct administration of asolution of the polypeptide. It is preferred that a sustained release bea release which occurs over a period of at least about one week, such asat least about two weeks, at least about three weeks or at least aboutfour weeks. The sustained release can be a continuous or a discontinuousrelease, with relatively constant or varying rates of release. Thecontinuity of release and level of release can be affected by the typeof polymer composition used (e.g., monomer ratios, molecular weight,block composition, and varying combinations of polymers), polypeptideloading, and/or selection of excipients to produce the desired effect.

[0062] As used herein, a therapeutically effective amount,prophylactically effective amount or diagnostically effective amount isthe amount of the sustained release composition needed to elicit thedesired biological response following administration.

[0063] Polymers suitable to form the sustained release composition ofthis invention are biocompatible polymers which can be eitherbiodegradable or non-biodegradable polymers or blends or copolymersthereof. A polymer is biocompatible if the polymer and any degradationproducts of the polymer are non-toxic to the recipient and also possessno significant deleterious or untoward effects on the recipient's body,such as an immunological reaction at the injection site.

[0064] Biodegradable, as defined herein, means the composition willdegrade or erode in vivo to form smaller units or chemical species.Degradation can result, for example, by enzymatic, chemical and physicalprocesses. Suitable biocompatible, biodegradable polymers include, forexample, poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,polyesteramides, polyanydrides, poly(amino acids), polyorthoesters,poly(dioxanone)s, poly(alkylene alkylate)s, copolymers or polyethyleneglycol and polyorthoester, biodegradable polyurethane, blends thereof,and copolymers thereof.

[0065] Suitable biocompatible, non-biodegradable polymers includenon-biodegradable polymers selected from the group consisting ofpolyacrylates, polymers of ethylene-vinyl acetates and other acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinylchloride, polyvinyl flouride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blendsthereof, and copolymers thereof.

[0066] Acceptable molecular weights for polymers used in this inventioncan be determined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, end group chemistry andrate of dissolution of polymer in solvent. Typically, an acceptablerange of molecular weight is of about 2,000 Daltons to about 2,000,000Daltons. In a preferred embodiment, the polymer is biodegradable polymeror copolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide)(hereinafter “PLG”) with a lactide:glycolideratio of about 1:1 and a molecular weight of about 5,000 Daltons toabout 70,000 Daltons. In an even more preferred embodiment, themolecular weight of the PLG used in the present invention has amolecular weight of about 5,000 Daltons to about 50,000 Daltons.

[0067] The sustained release composition of this invention can be formedinto many shapes such as a film, a pellet, a cylinder, a disc or amicroparticle. A microparticle, as defined herein, comprises a polymercomponent having a diameter of less than about one millimeter and havingbiologically active polypeptide dispersed or dissolved therein. Amicroparticle can have a spherical, non-spherical or irregular shape.Typically, the microparticle will be of a size suitable for injection. Atypical size range for microparticles is 1000 microns or less. In aparticular embodiment, the microparticle ranges from about one to about180 microns in diameter.

[0068] Bioavailability, as that term is used herein, refers to theamount of therapeutic that reaches the general circulation. That is, thecalculated Area Under the Curve (AUC) for the release profile of aparticular polypeptide during the time period starting at postadministration and ending at a predetermined time point. As isunderstood in the art, the release profile is generated by graphing theserum levels of a biologically active agent in a subject (Y-axis) atpredetermined time point (X-axis). Bioavailability is often referred toin terms of % Bioavailability, which is the bioavailablity achieved fora particular polypeptide following administration of a sustained releasecomposition divided by the bioavailability achieved for a particularpolypeptide following administration of the same dose of drugintravenously multiplied by 100.

[0069] Increased bioavailability as that term is used herein refers toan increase in the bioavailability of a biologically active polypeptideagent from a sustained release composition when coadministered with acorticosteroid in comparison to the administration in the absence ofcorticosteroid over a time period beginning post administration andending at the targeted timepoint for the particular formulation.

[0070] A modification of the release profile can be confirmed byappropriate pharmacokinetic monitoring of the patient's serum for thepresence of the biologically active polypeptide agent. For example,specific antibody-based testing (e.g., ELISA and IRMA), as is well knownin the art, can be used to determine the concentration of certainbiologically active polypeptide agents in the patient's serum. Anexample of such testing is described herein for exendin-4.

[0071] Pharmacodynamic monitoring of the patient to monitor thetherapeutic effects of the agent upon the patient can be used to confirmretention of the biologically activity of the released agent. Methods ofmonitoring pharmacodynamic effects can be selected based upon thebiologically active polypeptide agent being administered using widelyavailable techniques.

[0072] A number of methods are known by which sustained releasecompositions (polymer/biologically active polypeptide matrices) of theinvention can be formed. In many of these processes, the material to beencapsulated is dispersed in a solvent containing a wall formingmaterial (e.g., biocompatible polymer). At a single stage of theprocess, solvent is removed and thereafter the microparticle product isobtained.

[0073] Methods for forming a composition for the sustained release ofbiologically active polypeptide agent are described in U.S. Pat. No.5,019,400, issued to Gombotz et al., and issued U.S. Pat. No. 5,922,253issued to Herbert et al. the teachings of which are incorporated hereinby reference in their entirety.

[0074] In this method, a mixture comprising a biologically activepolypeptide, a biocompatible polymer and a polymer solvent is processedto create droplets, wherein at least a significant portion of thedroplets contains polymer, polymer solvent and the biologically activeagent. These droplets are then frozen by a suitable means. Examples ofmeans for processing the mixture to form droplets include directing thedispersion through an ultrasonic nozzle, pressure nozzle, Rayleigh jet,or by other known means for creating droplets from a solution.

[0075] Means suitable for freezing droplets include directing thedroplets into or near a liquified gas, such as liquid argon or liquidnitrogen to form frozen microdroplets which are then separated from theliquid gas. The frozen microdroplets are then exposed to a liquid orsolid non-solvent, such as ethanol, hexane, ethanol mixed with hexane,heptane, ethanol mixed with heptane, pentane or oil.

[0076] The solvent in the frozen microdroplets is extracted as a solidand/or liquid into the non-solvent to form a polymer/active agent matrixcomprising a biocompatible polymer and a biologically active agent.Mixing ethanol with other non-solvents, such as hexane, heptane orpentane, can increase the rate of solvent extraction, above thatachieved by ethanol alone, from certain polymers, such aspoly(lactide-co-glycolide) polymers.

[0077] A wide range of sizes of sustained release compositions can bemade by varying the droplet size, for example, by changing theultrasonic nozzle diameter. If the sustained release composition is inthe form of microparticles, and very large microparticles are desired,the microparticles can be extruded, for example, through a syringedirectly into the cold liquid. Increasing the viscosity of the polymersolution can also increase microparticle size. The size of themicroparticles which can be produced by this process ranges, forexample, from greater than about 1000 to about 1 micrometers indiameter.

[0078] Yet another method of forming a sustained release composition,from a suspension comprising a biocompatible polymer and a biologicallyactive agent, includes film casting, such as in a mold, to form a filmor a shape. For instance, after putting the suspension into a mold, thepolymer solvent is then removed by means known in the art, or thetemperature of the polymer suspension is reduced, until a film or shape,with a consistent dry weight, is obtained.

[0079] A further example of a conventional microencapsulation processand microparticles produced thereby is disclosed in U.S. Pat. No.3,737,337, incorporated by reference herein in its entirety, wherein asolution of a wall or shell forming polymeric material in a solvent isprepared. The solvent is only partially miscible in water. A solid orcore material is dissolved or dispersed in the polymer-containingmixture and, thereafter, the core material-containing mixture isdispersed in an aqueous liquid that is immiscible in the organic solventin order to remove solvent from the microparticles.

[0080] Another example of a process in which solvent is removed frommicroparticles containing a substance is disclosed in U.S. Pat. No.3,523,906, incorporated herein by reference in its entirety. In thisprocess a material to be encapsulated is emulsified in a solution of apolymeric material in a solvent that is immiscible in water and then theemulsion is emulsified in an aqueous solution containing a hydrophiliccolloid. Solvent removal from the microparticles is then accomplished byevaporation and the product is obtained.

[0081] In still another process as shown in U.S. Pat. No. 3,691,090,incorporated herein by reference in its entirety, organic solvent isevaporated from a dispersion of microparticles in an aqueous medium,preferably under reduced pressure.

[0082] Similarly, the disclosure of U.S. Pat. No. 3,891,570,incorporated herein by reference in its entirety, shows a method inwhich solvent from a dispersion of microparticles in a polyhydricalcohol medium is evaporated from the microparticles by the applicationof heat or by subjecting the microparticles to reduced pressure.

[0083] Another example of a solvent removal process is shown in U.S.Pat. No. 3,960,757, incorporated herein by reference in its entirety.

[0084] Tice et al., in U.S. Pat. No. 4,389,330, describe the preparationof microparticles containing an active agent by a method comprising: (a)dissolving or dispersing an active agent in a solvent and dissolving awall forming material in that solvent; (b) dispersing the solventcontaining the active agent and wall forming material in acontinuous-phase processing medium; (c) evaporating a portion of thesolvent from the dispersion of step (b), thereby forming microparticlescontaining the active agent in the suspension; and (d) extracting theremainder of the solvent from the microparticles.

[0085] A suitable polymer solution contains between about 1% (w/v) andabout 30% (w/v) of a suitable biocompatible polymer, wherein thebiocompatible polymer is typically dissolved in a suitable polymersolvent. Preferably, a polymer solution contains about 2% (w/v) to about20% (w/v) polymer.

[0086] A suitable polymer solvent, as defined herein, is a solvent inwhich the polymer is soluble, in which some, none, or all of the othercomponents are soluble and in which the other components arenon-reactive. Examples of suitable polymer solvents include polarorganic liquids, such as methylene chloride, chloroform, ethyl acetate,methyl acetate, hexafluoroisopropanol, acetone, dimethylsulfoxide andcombinations thereof.

[0087] Further, excipients can be present in the sustained releasecomposition. The excipients can be added to maintain the potency of thebiologically active polypeptide agent over the duration of releaseand/or modify polymer degradation. Suitable excipients include, forexample, buffer salts, carbohydrates, metal cations, amino acids, fattyacids, surfactants, and bulking agents, and are known to those skilledin the art. An acidic or a basic excipient is also suitable. The amountof excipient used is based on ratio to the biologically activepolypeptide agent, on a weight basis and can be determined by one ofskill in the art using available methods.

[0088] Buffer salt, as defined herein is the salt remaining followingremoval of solvent from a buffer. Buffers are solutions containingeither a weak acid and a related salt of the acid, or a weak base and asalt of the base. Buffers can maintain a desired pH to assist instabilizing the formulation. This maintenance of pH can be affordedduring processing, storage and/or release. For example, the buffer canbe monobasic phosphate salt or dibasic phosphate salt or combinationsthereof or a volatile buffer such as ammonium bicarbonate. Other buffersinclude, but are not limited to, acetate, citrate, succinate and aminoacids such as glycine, arginine and histidine. The buffer when presentin the final sustained release composition can range from about 0.01% toabout 10% of the total weight. In a preferred embodiment, the buffersalt is a sodium acetate salt.

[0089] A surfactant can be present in the sustained release composition.The surfactant can act to further modify release of the biologicallyactive polypeptide from the polymer matrix, or can act to furtherstabilize the biologically active polypeptide or a combination thereof.The presence of surfactant can in some instances assist in minimizingadsorption of the biologically active polypeptide to the biocompatiblepolymer. The amount of surfactant present in the sustained releasecomposition can range from about 0.1% w/w to about 50% w/w of the dryweight of the composition.

[0090] Surfactants, as the term is used herein refers to any substancewhich can reduce the surface tension between immiscible liquids.Suitable surfactants which can be added to the sustained releasecomposition include polymer surfactants, such as nonionic polymersurfactants, for example, poloxamers, polysorbates, polyethylene glycols(PEGs), polyoxyethylene fatty acid esters, polyvinylpyrrolidone andcombinations thereof. Examples of poloxamers suitable for use in theinvention include poloxamer 407 sold under the trademark PLURONIC® F127,and poloxamer 188 sold under the trademark PLURONIC® F68, both availablefrom BASF Wyandotte. Examples of polysorbates suitable for use in theinvention include polysorbate 20 sold under the trademark TWEEN® 20 andpolysorbate 80 sold under the trademark TWEEN® 80. Cationic surfactants,for example, benzalkonium chloride, are also suitable for use in theinvention. In addition, bile salts, such as deoxycholate andglycocholate are suitable as surfactants based on their highly effectivenature as detergents.

[0091] Bulking agents typically comprise inert materials. Suitablebulking agents are known to those skilled in the art.

[0092] A metal cation component which is dispersed within the polymermatrix can also be used as an optional excipient. This metal cationcomponent acts to further modulate the release of polypeptide and is notcomplexed with the polypeptide. A metal cation component used inmodulating release typically comprises at least one type of multivalentmetal cation. Examples of metal cation components suitable to modulateprotein release include or contain, for example, Mg(OH)₂, MgCO₃ (such as4MgCO₃.Mg(OH)₂.5H₂O), Zn(OAc)₂, ZnSO₄ and ZnCl₂. A suitable ratio ofmetal cation component to polymer is between about 1:99 to about 1:2 byweight. The optimum ratio depends upon the polymer and the metal cationcomponent utilized. A polymeric matrix containing a dispersed metalcation component to modulate the release of a biologically active agentfrom the polymeric matrix is further described in U.S. Pat. No.5,656,297 and co-pending U.S. patent application Ser. No. 08/727,531,the teachings of both of which are incorporated herein by reference intheir entirety.

[0093] Detailed procedures for microparticle formation are set forth inthe Working Examples.

[0094] Without being bound by a particular theory it is believed thatthe release of the biologically active polypeptide, can occur by twodifferent mechanisms. First, the biologically active polypeptide can bereleased by diffusion through aqueous filled channels generated in thepolymeric matrix, such as by the dissolution of the polypeptide, or byvoids created by the removal of the polymer solvent during thepreparation of the sustained release composition. A second mechanism isthe release of the polypeptide due to degradation of the polymer. Therate of degradation can be controlled by changing polymer propertiesthat influence the rate of hydration of the polymer. These propertiesinclude, for instance, the ratio of different monomers, such as lactideand glycolide, comprising a polymer; the use of the L-isomer of amonomer instead of a racemic mixture; and the molecular weight of thepolymer. These properties can affect hydrophilicity and crystallinity,which control the rate of hydration of the polymer. By altering theproperties of the polymer, the contributions of diffusion and/or polymerdegradation to release can be controlled. For example, increasing theglycolide content of a poly(lactide-co-glycolide) polymer and/ordecreasing the molecular weight of the polymer can enhance thehydrolysis of the polymer and thus, provides an increased rate ofrelease of protein due to polymer erosion.

[0095] In addition, the rate of polymer hydrolysis is increased innon-neutral pH. Therefore, an acidic or a basic excipient such as aninorganic acid or inorganic base can be added to the polymer solution,used to form the microparticle, to alter the polymer erosion rate

[0096] The composition of this invention can be administered to a human,or other animal, by injection, implantation (e.g., subcutaneously,intramuscularly, intraperitoneally, intracranially, and intradermally),administration to mucosal membranes (e.g., intranasally, intravaginally,intrapulmonary or by means of a suppository), or in situ delivery (e.g.,by enema or aerosol spray) to provide the desired dosage of biologicallyactive polypeptide based on the known parameters for treatment with suchprotein of the various medical conditions.

[0097] The sustained release composition can be administered using anydosing schedule which achieves the desired therapeutic levels for thedesired period of time. For example, the sustained release compositioncan be administered and the patient monitored until levels of the drugbeing delivered return to baseline. Following a return to baseline, thesustained release composition can be administered again. Alternatively,the subsequent administration of the sustained release composition canoccur prior to achieving baseline levels in the patient.

[0098] For example, when the sustained release composition hasincorporated therein a hormone, particularly an anti-diabetic orglucoregulatory peptide, for example, GLP-1, GLP-2, exendin-3, exendin-4or agonists, analogs or derivatives thereof and the composition isadministered in a therapeutically effective amount to treat a patientsuffering from diabetes mellitus, impaired glucose tolerance (IGT),obesity, cardiovascular (CV) disorder or any other disorder that can betreated by one of the above polypeptides or derivatives, analogs oragonists thereof.

[0099] Other conditions which can be treated by administering thesustained release composition of the invention include Type I and TypeII diabetes which can be treated with sustained release compositionhaving insulin incorporated therein. In addition, when the incorporatedpolypeptide is FSH or analogs thereof the sustained release compositioncan be used to treat infertility. In other instances, the sustainedrelease composition can be used to treat Multiple Sclerosis when theincorporated polypeptide is beta interferon or a mutein thereof. As canbe realized, the sustained release composition can be to treat diseasewhich respond to administration of a give polypeptide.

[0100] Even though the invention has been described with a certaindegree of particularity, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing disclosure. Accordingly, it is intendedthat all such alternatives, modifications, and variations which fallwithin the spirit and scope of the invention be embraced by the definedclaims.

[0101] The invention will now be further and specifically described bythe following examples.

[0102] Exemplifications

[0103] Microparticle Preparation

[0104] The sustained release compositions described herein were preparedby any of emulsion, coacervation, and cryogenic microencapsulationtechniques. The general process associated with each technique isdescribed below.

[0105] Coacervation—W/O/O Process

[0106] The coacervation process, also referred to herein as awater-oil-oil (W/O/O) process, requires formation of a water-in-oilemulsion with aqueous drug and organic polymer solutions. An oil,typically a silicone oil, was then added to the water-in-oil emulsion toinduce phase separation and to precipitate the polymer. The embryonicmicroparticles were then quenched in a solvent that removes the oil andpolymer solvent.

[0107] Exendin-4 was encapsulated in PLG polymer using a water-oil-oil(W/O/O) emulsion system. The initial embryonic microparticles wereformed in a W/O/O inner emulsion step after which they were subjected tocoacervation and hardening steps. The microparticles were collected,dried and filled into vials. Further details of each step in thecomplete process is set forth below.

[0108] Inner Emulsion Formation

[0109] A water-in-oil emulsion was created using sonication. The waterphase of the emulsion contained dissolved exendin-4 and variousexcipients in water. Typically, sucrose and ammonium sulfate werepresent as excipients but other excipients and combinations ofexcipients were investigated. The PLG phase contained polymer dissolvedin methylene chloride.

[0110] Coacervation Formation

[0111] Coacervation was induced by adding silicone oil at a controlledrate to the inner emulsion with agitation, forming embryonicmicroparticles. The embryonic microparticles formed were relatively softand required hardening.

[0112] Microparticle Hardening

[0113] The embryonic microparticles were added to a heptane/ethanolsolvent mixture with gentle agitation. The solvent mixture hardened theembryonic microparticles. After hardening for about one hour at about 3°C., the solvent mixture was decanted and pure heptane was added at 3° C.and mixed for about one hour.

[0114] Microparticles Drying and Collection

[0115] After the hardening step, the microparticles were transferred andcollected on a fine mesh pore-plate inside a drying chamber. A finalheptane rinse of the hardening vessel was performed. The microparticleswere dried with nitrogen gas over a four-day period with temperatureramping from about 3° C. to about 38° C.

[0116] In general, PLG was dissolved in methylene chloride. The innerwater phase was prepared by dissolving the exendin-4, sucrose andammonium sulfate in water or an aqueous buffer. The aqueous solution wasthen injected into the polymer solution while probe sonicating. Theresultant water/oil emulsion was then added to an emulsion reactor.Silicone oil (350 centiStokes) was slowly added to the reactor viaperistaltic pump with stirring at about 1000 rpm. The mixture was thenadded to n-heptane. After stirring for about two hours, themicroparticles were isolated by filtration and vacuum dried overnight.

[0117] Emulsion Process—W/O/W Process

[0118] The emulsion process is also referred to as a water-oil-water(W/O/W) process. Briefly, an aqueous solution of drug was dispersed in apolymer solution which was then emulsified in an outer aqueous phase(e.g., PVA). The microparticles were then hardened in an aqueous quench.

[0119] In a typical experiment, PLG (1.96 g) was dissolved in methylenechloride (22.5 g) and drug (e.g., exendin-4) was dissolved in water (20mg exendin-4 in 1.75 g water). The drug solution was then drawn up in asyringe and injected into the polymer solution while it was probesonicated. The resultant W/O emulsion was then quickly added to anemulsion reactor containing 125 g aqueous 5% polyvinyl alcohol (PVA).The stir rate of the reactor was set to about 800 RPM. The mixture wasstirred for about 1.5 minutes and then added to a water quench (2.8 L at10° C.). After about two hours in the quench, the hardenedmicroparticles were isolated by filtration and vacuum dried overnight.

[0120] Cryogenic Process

[0121] The cryogenic process used atomization to form droplets ofpolymer solution containing drug. Embryonic microparticles were thenfrozen in liquid nitrogen and the polymer solvent was removed through asubsequent ethanol extraction technique.

[0122] The cryogenic processing to produce microparticles included twosteps: (1) the production of a lyophilizate; and (2) microencapsulationusing a low-temperature, non-aqueous technique. Lyophilizates wereformulated by atomizing a mixture of drug and excipient using atwo-fluid nozzle, freezing the atomized droplets and drying the frozendroplets using lyophilization. It is understood that any suitablemethods of drying known in the art can be employed. Specifically, frozendroplets were dried for about 7 days at a primary drying condition of−26° C. shelf and 96 mTorr chamber pressure followed by secondary dryingfor an additional 3 days at about 20° C. and 0 mTorr.

[0123] Exendin-4 containing microparticles were produced with thecryogenic, non-aqueous process, all at a nominal target load of 1% drug.Lyophilizates were suspended in an organic solution consisting of 6% 4APLG dissolved in methylene chloride. This suspension was sonicated forabout 4 minutes on ice, and then the suspension was atomized using asonication nozzle and frozen by contacting with liquid nitrogen layeredover a bed of frozen ethanol. The sample was warmed to −80° C. in orderto allow for microparticle hardening and extraction of solvent. Themicroparticles were then filtered and dried.

[0124] Solid/Oil/Water (S/O/W) and Solid/Oil/Oil (S/O/O)Processes

[0125] Solid drug was also encapsulated using modified versions of theemulsion and coacervation processes described above. These modifiedprocesses are referred to solid/oil/water (SO/W) and solid/oil/oil(S/O/O).

[0126] For example, solid exendin-4 was suspended in methylene chloridecontaining 6% PLG and sonicated for about four minutes on ice.Subsequent processing was conducted in a manner analogous to either theW/O/O or W/O/W methods.

[0127] Polymer:

[0128] Examples of specific PLG polymers suitable for use are listedbelow. All of the polymers employed in the following examples are setforth in the list and all listed polymers were purchased from Alkernes,Inc. of Cincinnati, Ohio and can be described as follows:

[0129] Polymer 2A: Poly(lactide-co-glycolide); 50:50 lactide:glycolideratio; 12.3 kD Mol. Wt.; IV=0.15 (dL/g).

[0130] Polymer 2A-1: Poly(lactide-co-glycolide); 65:35 lactide:glycolideratio; 16 kD Mol. Wt.; IV=0.19 (dL/g).

[0131] Polymer 2.5A: Poly(lactide-co-glycolide); 50:50 lactide:glycolideratio; 25 kD Mol. Wt.; IV=0.24 (dL/g).

[0132] Polymer 3A: Poly(lactide-co-glycolide); 50:50 lactide:glycolideratio; 47 kD Mol. Wt.; IV=0.38 (dL/g).

[0133] Polymer 3.5A: Poly(lactide-co-glycolide); 50:50 lactide:glycolideratio; Mol. Wt., Not Determined; IV=0.42 (dL/g).

[0134] Polymer 4A: Poly(lactide-co-glycolide); 50:50 lactide:glycolideratio; Mol. Wt. 45-64 kD; IV=0.45-0.47 (dL/g).

[0135] Polymer 4A-1: Poly(lactide-co-glycolide); 65:35 lactide:glycolideratio; Mol. Wt. 53 kD; IV=0.43 (dL/g).

[0136] PURIFICATION OF PLG: It is known in the art (See, for example,Peptide Acylation by Poly(α-Hydroxy Esters) by Lucke et al.,Pharmaceutical Research, Vol. 19, No. 2, p. 175-181, February 2002) thatproteins and peptides which are incorporated in PLG matrices can beundesirably altered (e.g., degraded or chemically modified) as a resultof interaction with degradation products of the PLG or impuritiesremaining after preparation of the polymer. As such, the PLG polymersused in the preparation of the majority of microparticle formulationsdescribed herein, were purified prior to preparation of the sustainedrelease compositions using art recognized purification methods.

[0137] Methods of Analysis

[0138] The following methods were employed to analyze samples producedduring the production and characterization of the microparticlesdescribed herein. For example, the bulk polypeptide can be analyzed,lyophilizates of the polypeptide can be analyzed, polypeptide extractedfrom the microparticles can be analyzed, as well as polypeptide releasedfrom the microparticles.

[0139] Exendin-4 Analysis

[0140] Analytical tests for purity, quantitation and identification ofexendin-4 are as follows.

[0141] Reversed Phase-HPLC (RP-HPLC)

[0142] RP-HPLC was used to separate native exendin-4 from oxidized andtruncated peptide. The relative amount of native exendin was reported asa percent of total absorption by UV; both 214 nm and 280 m can be usedfor this purpose. The total peak area of all UV absorption impuritieswas used to define percent (%) purity of native exendin-4 and anyimpurities ≧0.1% of total AUC. Impurities are defined by their relativeretention time compared to native AC2993 (RRT=1.00).

[0143] Samples were injected onto a C18 column (150 mm×4.6 mm),temperature-controlled at 40° C. The method was performed using gradientconditions with water and acetonitrile acidified with 0.1% TFA. The runtime for the method was about 40 minutes per injection. The method wasused to determine drug integrity.

[0144] Microsphere Characterization

[0145] Exendin-4 microspheres were routinely characterized with respectto drug content, particle size, residual solvents, initial in vitrorelease, and PK characteristics in rats. Drug was extracted to obtain apreliminary assessment of exendin-4 purity post-encapsulation inselected batches.

[0146] Particle Size Determination

[0147] The particle size distributions of the microparticle formulationsdescribed herein were determined with a Coulter LS 230 instrumentemploying laser diffraction technique equipped with the micro volumemodule. Generally, microparticles were suspended in an appropriatevehicle and subjected to analysis according to manufacturer'sinstructions. Mass median diameter, 10% greater than (microns), and 90%greater than (microns) were determined.

[0148] Load of Exendin-4

[0149] The exendin-4 content of bulk microparticles was determined bytotal nitrogen analysis using in an Exeter Analytical, model 240XA orXAI 240 nitrogen analyzer. Samples (5 mg) were combusted at 980° C. toproduce carbon dioxide, nitrogen, and nitrogen oxides. The amount ofexendin-4 present was determined based on the nitrogen content and thetheoretical nitrogen content of exendin-4 (based on chemical formula ofexendni-4, C₁₈₄H₂₈₂N₅₀O₆₀S).

[0150] Alternatively, following extraction, exendin-4 was quantifiedusing Size-Exclusion Chromatograpy (SEC). The method uses a silica-basedTSK-GEL SW column, isocratic elution with 0.1% TFA in water/acetonitrilemobile phase, and UV absorption at 280 nm

[0151] Size Exclusion Chromatography (SEC) was employed to quantify drugload following extraction from the microparticles. SEC was conducted ona silica-based TSK-GEL SW column available from Tosoh Biosep (Cat. #:08540). The flow rate was 0.8 mL/min with a column load of about 10 μLcontaining about 0.2 mg/mL drug and a detector setting of 280 nm. Theelution system was isocratic using 0.1% TFA in water/acetonitrile mobilephase.

[0152] Exendin-4 was extracted from the microparticles and the polymerwas removed prior to determining the integrity, purity and identity ofexendin-4. Briefly, the microparticles were dissolved in methylenechloride and the extracted exendin-4 was collected on a 0.1 μm filter,dried and then reconstituted in acetate buffer.

[0153] Residual Solvents

[0154] A single method was used for quantitation of heptane andmethylene chloride. The equipment consisted of an HP 5890 Series 2 gaschromatograph with an Rtx 1301, 30 m×0.53 mm column. About 130 mgmicroparticles were dissolved in 10 ml N,N-dimethylformamide. Propylacetate was used as the internal standard. The sample preparation wasadjusted so that concentrations of methylene chloride as low as 0.03%can be quantitated.

[0155] In Vitro Initial Release

[0156] The initial release of exendin-4 was determined by measuring theconcentration of exendin-4 after 24 hours. Typically, microparticleswere incubated in isotonic, physiologic media (pH 7.4) at about 37° C.for about 24 hours. Exendin-4 concentration was determined usingappropriate methods such as SEC and RP-HPLC. In vitro initial release ofabout 0.1% can be quantitated employing such methods.

[0157] Animal Studies

[0158] All pharmacokinetic (PK) studies described herein were conductedin adult male Sprague-Dawley rats weighing approximately 450±50 g (age12-13 weeks).

[0159] For I.V. bolus studies, all animals were fully anesthetized with5% halothane and a skin incision was made superior to the jugular vein.A dose of 30 μg of exendin-4 per rat was administered into the jugularvein and the incision was closed with wound clips. Blood samples werecollected via lateral tail vein 5, 10, 20, 30, 60, 90, 120, 150, 180,240, 300 and 360 minutes post-dose.

[0160] For PK characterization of the microparticle formulations, eachanimal received a subcutaneous injection of microparticles suspended indiluent (3% carboxymethylcellulose, 0.9% NaCl, 0.1% Tween 20) to theinter-scapular region. Generally, the dose was approximately 0.9 mgexendin-4 per rat and the injection volume was 0.75 mL. Blood sampleswere collected via lateral tail vein at 2, 4, 6, 10, 24 hours post dose,and typically at 2, 4, 7, 19, 14, 17, 21, 24 and 28 days post-dose.Blood samples were immediately placed in MICROTAINER® tubes containingEDTA and centrifuged at about 14,000×g for about two minutes. Plasma wasthen transferred to MICROTAINER® tubes without additive and stored at−70° C. until time of assay. IRMA was used to determine plasma exendinconcentrations.

[0161] In Vivo Release-IRMA

[0162] The method for quantifying exendin-4 in plasma is a sandwichimmunoassay, with the analyte captured by a solid phase monoclonalantibody EXE4:2-8.4 and detected by the radioiodinated monoclonalantibody GLP-1:3-3. Counts bound are quantitated from a standardcalibration curve. This assay is specific for exendin-4 and does notdetect exendin-4 (3-39) a major metabolite or GLP-1. A typical standardcurve range is 30 pg/mL to 2000 pg/mL depending on the age of the tracerantibody.

[0163] Microparticle Characteristics

[0164] For all microparticle formulations which were prepared herein, aW/O/O process was employed. The inner phase was either water or sodiumacetate buffer at a selected concentration and pH. The amount ofexendin-4 and excipients present in the prepared formulations isexpressed as a % (w/w) based on the final weight of the sustainedrelease composition. The % (w/w) is a nominal percentage, except wereindicated.

[0165] Discovery of Initial Formulations (IF)

[0166] As a result significant research, it was discovered that InitialFormulations 1-4 in Table 1 (IF-1, IF-2, IF-3 and IF-4) provided adesired release profile for exendin-4. For example, the InitialFormulations achieved a release of therapeutic levels of exendin-4 forabout 3-4 weeks with minimal initial release. The Initial Formulationswere prepared using the W/O/O process described above. The inner wateremulsion containing exendin-4 was formulated as follows: 50 mg/mLexendin-4, 30 mM sodium acetate buffer pH 4-4.5, and 50 mg/mL of sucroseor mannitol. The polymer phase consisted of a 3A or 4A (50:50,lactide:glycolide) PLG as described above. The nominal load of-exendin-4 for IF-1 through IF-4 was 1% w/w with a nominal load of sugar(mannitol or sucrose) at 1% w/w. TABLE 1 % EXENDIN-4 IN DRUG PHASEFORMULATION % EXCIPIENT IN INITIAL (INNER AQUEOUS POLYMER (W/W*,FORMULATION FORMULATION EMULSION) PHASE NOMINAL) (W/W, NOMINAL) IF-1 50mg/mL 3A 50:50 1% 1% Sucrose Exendin-4 50 mg/mL sucrose 30 mM sodiumacetate (pH 4-4.5) IF-2 50 mg/mL 4A 50:50 1% 1% Sucrose Exendin-4 50mg/mL sucrose 30 mM sodium acetate (pH 4-4.5) IF-3 50 mg/mL 3A 50:50 1%1% Mannitol Exendin-4 50 mg/mL mannitol 30 mM sodium acetate (pH 4-4.5)IF-4 50 mg/mL 4A 50:50 1% 1% Mannitol Exendin-4 50 mg/mL mannitol 30 mMsodium acetate (pH 4-4.5)

[0167] The release profiles for Initial Formulations 1-4 of Table 1 aredepicted in FIG. 1.

[0168] Initial Formulation Development

[0169] Development of the Initial Formulations IF-1-IF-4 in Table 1 wasbased on achieving low initial release of exendin-4 coupled withsustained plasma levels of exendin-4. As such, the effects of variousformulation and process parameters were examined. The parametersinvestigated included: encapsulation process, drug loading, polymercharacteristics, drug formulation and excipients.

[0170] Polymer

[0171] The polymers employed in the development of the InitialFormulations are found in the list of polymers described above and werechosen to provide a release from about 3 weeks to about 3 months. As canbe seen in Table 1, the Initial Formulations employed a 3A (50:50) and4A (50:50) PLG. The Initial Formulations were found to release exendin-4for about 3-4 weeks following administration.

[0172] Drug Loading

[0173] The drug loading in the formulations employed in the developmentof the Initial Formulations ranged from about 1% to about 5%. The drugloading refers to the nominal drug load and is a % (w/w) based on thefinal weight of the sustained release composition. For exendin-4, thehigh potency is amenable to drug loading in this range. As can be seenin Table 1 drug loading for the Initial Formulations was set at 1%.

[0174] Formulation of Drug

[0175] The majority of the development work resulting in the InitialFormulations was conducted with the exendin-4 in aqueous solutions. Thesolutions ranged in concentration from about 10 to 150 mg of exendin-4per mL of water.

[0176] Initially, exendin-4 was encapsulated from an aqueous solutionalone (no excipients). Bioavailability of these formulations wasvariable however leading to studies aimed at determining potentialstabilizing excipient for exendin-4. Exendin-4 stability was determinedas a function of pH and concentration. As a result, formulations wereprepared using acetate buffered solution at pH 4. Details of this studyare set forth below in Example 1.

[0177] Formulations containing sucrose, ammonium sulfate or Tween −20 inthe aqueous phase were also evaluated. Details of this study are setforth in Example 2.

EXAMPLE 1 Effect of pH and Concentration on the Integrity ofUnencapsulated Exendin-4 (pH 7)

[0178] The effect of pH and concentration on the integrity of exendin-4was investigated. The studies were conducted at 37° C. At pH 7 there wasa dramatic loss of % native exendin-4 at the lowest concentration tested(0.3 mg/mL). The rate of loss was decreased as the concentration wasincreased to 12.5 mg/mL and at 50 mg/mL stability was further improved.The results are depicted graphically in FIG. 2A.

EXAMPLE 2 Effect of pH and Concentration on the Integrity ofUnencapsulated Exendin-4 (pH 4)

[0179] The same series of exendin-4 concentrations as described inExample 1 were conducted at pH 4. Good stability was observed over theentire concentration range up to 49 days of incubation. The results oftesting at pH 4 are set forth in FIG. 2B.

EXAMPLE 3 Effect of Excipients on the Integrity of UnencapsulatedExendin-4

[0180] The effect of adding various excipients to exendin-4 formulatedat 50 mg/mL and pH 4 was examined. The excipients tested were sucroseand ammonium sulfate, both at at a 1:1 weight ratio with the exendin-4(i.e., 50 mg/mL) and 0.1% TWEEN-20.

[0181] As can be seen in FIG. 3. there was further moderate improvementin exendin-4 stability in the presence of the excipients.

EXAMPLE 4 Microparticle Development

[0182] A series of microparticle batches were prepared using the W/O/Oprocess described above. A description of each microparticle formulationis set forth in Table 2. Briefly, the effect of exendin-4 concentrationin the inner aqueous phase (12.5 mg/mL or “high water” and 50 mg/mL or“low water”) and the addition of sucrose or ammonium sulfate (at anequal weight relative to exendin) to the inner aqueous phase wasexamined. In all cases the inner aqueous phase contained 30 mM acetatebuffer at pH 4. TABLE 2 % EXENDIN-4 % EXCIPIENT MICROPARTICLE INFORMULATION IN FORMULATION DRUG Cmax BIOAVAILABILITY LOT (W/W) (W/W)POLYMER PHASE (pg/mL) (%) M-1 1% 0% 4A 50 mg/mL, 383 ± 240 8 pH = 4 M-21% 0% 4A 12.5 mg/mL 855 ± 127 4 pH = 4 M-3 1% 1% 4A 50 mg/mL, 1366 ±1860 15 SUCROSE pH = 4, sucrose M-4 1% 1% 4A 12.5 mg/mL, 988 ± 170 10SUCROSE pH = 4, sucrose M-5 1% 1% 4A 50 mg/mL, 15371 ± 8597  45 AMMONIUMpH = 4, SULFATE (NH₄)₂SO₄ M-6 1% 1% 4A 12.5 mg/mL, 10839 ± 875  60AMMONIUM pH = 4, SULFATE (NH₄)₂SO₄

[0183] The release profiles for Formulations M-1, M-2, M-4 and M-6 areset forth in FIG. 4. The release profile shows that the low waterformulation had a lower Cmax, the addition of sucrose resulted in a lesspronounced lag phase following the initial release and generally higherlevels of exendin-4 for about the first two weeks, and the ammoniumsulfate-containing batch exhibited a large initial release.

EXAMPLE 5 Additional Microparticle Formulations

[0184] Additional microparticles formulations were prepared indeveloping the Initial Formulations. All of the microparticleformulations were prepared using a W/O/O process. Polymer type waseither 3A or 4A PLG 50:50 described above. The load of exendin-4,sucrose and water were varied. In addition, mannitol as an excipient wasinvestigated. A description of each microparticle formulation is setforth in Table 3. TABLE 3 % EXENDIN-4 % EXCIPIENT IN IN MICROPARTICLEFORMULATION FORMULATION POLYMER DRUG Cmax BIOAVAILABILITY LOT (W/W)(W/W) PHASE PHASE (pg/mL) (%) M-7  1% 1% 4A 50 mg/mL, 1482 ± 640 18SUCROSE pH = 4, sucrose M-8  1% 1% 3A 50 mg/mL, 1794 ± 615 27 SUCROSE pH= 4, sucrose M-9  1% 2% 4A 50 mg/mL, 1087 ± 178 13 SUCROSE 0.2% pH = 4,MgCO₃ sucrose M-10 1% 2% 4A M-7 2607 ± 911 29 SUCROSE with 2 × sucroseM-11 2% 2% 4A M-7 1658 ± 522 18 SUCROSE with 2 × sucrose, exendin-4 andwater M-12 2% 2% 4A M-7 7691 ± 229 26 SUCROSE with 2 × sucrose andexendin-4 (100 mg/mL) M-13 1% 1% 4A M-7 1115 ± 687 18 MANNITOL withmannitol in place of sucrose M-14 1% 1% 3A M-7 1884 ± 820 22 MANNITOLwith mannitol in place of sucrose

[0185] The release profiles for Microparticle Formulations M-7, M-8 andM-9 are depicted graphically in FIG. 5. The release profiles forMicroparticle Formulations M-7, M10, M-11 and M-12 are depictedgraphically in FIG. 6. The release profile of Microparticle FormulationsM-13 and M-14 are depicted graphically in FIG. 7.

[0186] Initial Formulations—In Vivo

[0187] The Initial Formulations, IF-1, -2 and -4 of Table 1 were thenadministered to rats. The release profiles for these formulations aredepicted in FIG. 1 through 28 days post administration and in FIG. 8through 42 days post administration. Inspection of FIG. 8 shows thatIF-1, -2 and -4 provided an acceptable release profile in rats (15-32%bioavailability with a Cmax/Cave of about 2-3). Further research todiscover formulations with additional improvements in the releaseprofile of exendin-4 was then conducted resulting in the SelectedFormulations (SF) described below.

[0188] Such further formulation discovery was directed at increasingdrug load and determining excipients capable of further increasingbioavailability. Loads were examined in the range of 1% to 4%. Variousexcipients were tested including ammonium sulfate, acetate buffer,sugars, such as sucrose, mannitol and trehalose and the surfactant,Pluronic F-127.

[0189] Selected Formulations

[0190] The Selected Formulations resulting from the further formulationdiscovery, which were chosen for clinical testing, are set forth inTable 4. TABLE 4 % EXENDIN-4 DRUG PHASE IN FORMULATION % EXCIPIENT IN(INNER SELECTED W/W NOMINAL FORMULATION AQUEOUS POLYMER FORMULATION (W/WACTUAL) W/W EMULSION) PHASE SF-1   3% 2% SUCROSE, Exendin-4 (50 mg/mL 4A50:50 (2.5%) 0.3% in AMMONIUM water) SULFATE 2% sucrose, 0.3% ammoniumsulfate SF-2   3% 2% SUCROSE, Exendin-4 (50 mg/mL 4A 50:50 (2.8%) 0.5%in AMMONIUM water) SULFATE 2% sucrose, 0.5% ammonium sulfate

[0191] The sustained release profiles in rats for SF-1 (3% drug, 2%sucrose, 0.3% AS) and SF-2 (3% drug, 2% sucrose, 0.5% AS) are depictedgraphically in FIG. 9.

[0192] Additional Formulation Discovery

[0193] % LOAD VARIATION: Previously a 1% loading had been employed(Initial Formulations 1-4). Higher loads were tested to determine arange of drug loading for exendin-4. It was found that increasing theload from 1% to the range of about 2-3% was acceptable. Loads of 4% andhigher for exendin-4 resulted in an increased Cmax. The MicroparticleFormulations M15-M17 are described below and the sustained releaseprofile for each is depicted graphically in FIG. 10. M-15-M-17:

[0194] All microparticles contained 1:1 mannitol:drug (w:w) and 80 mMacetate buffer (pH=4.0) encapsulated in 4A, 50:50 PLG

[0195] M-15: 2% load

[0196] M-16: 3% load

[0197] M-17: 4% load

[0198] AMMONIUM SULFATE: The effect of ammonium sulfate on the releaseof exendin from microparticle formulations was further investigated.FIG. 11 shows that addition of 0.5% ammonium sulfate generally increasesexendin-4 plasma levels over the first two weeks. There was a dramaticincrease in bioavailability from about 12-15% to about 38-57%. Howeverthere was also an undesirable increase in the Cmax when ammonium sulfatewas used alone. The Microparticle Formulations depicted in FIG. 11 areset forth in Table 5. TABLE 5 % EXENDIN-4 IN DRUG PHASE FORMULATION %EXCIPIENT IN (INNER MICROPARTICLE W/W NOMINAL FORMULATION AQUEOUSPOLYMER LOT (W/W ACTUAL) W/W EMULSION) PHASE M-18 2% (1.89%^(#), 2%MANNITOL Exendin-4 4A 50:50 1.98%*) (50 mg/mL in 80 mM acetate buffer,pH = 4.0) 2% mannitol M-19 2% (1.79%*, 2% MANNITOL Exendin-4 (50 mg/mL4A 50:50 1.80%^(#)) in 80 mM acetate buffer, pH = 4.0) 2% mannitol M-202% (1.73%*, 2% MANNITOL Exendin-4 (50 mg/mL 4A 50:50 1.74%^(#)) 0.5% in80 mM AMMONIUM acetate SULFATE buffer, pH = 4.0) 2% mannitol, 0.5%ammonium sulfate M-21 2% (1.72%*, 2% MANNITOL Exendin-4 (50 mg/mL 4A50:50 1.75%^(#)) 0.5% AMMONIUM in 80 mM SULFATE acetate buffer, pH =4.0) 2% mannitol, 0.5% ammonium sulfate

[0199] SUGAR EXCIPIENT: Earlier formulations used sucrose and mannitolas the sugar excipient at a 1% load in the microparticle or a 1:1sugar:exendin-4 (w:w). In this further formulation development, sucrose,mannitol and trehalose, all at 2% load, were evaluated. In this study,the drug load was also 2% (therefore sugar-to-drug ratio 5 remained 1%)and the microparticles also had 0.5% ammonium sulfate present.

[0200] The sustained release profile for these formulation is set forthin FIG. 12. The Microparticle Formulations are as shown in Table 6.TABLE 6 % EXENDIN-4 IN % EXCIPIENT DRUG PHASE FORMULATION IN (INNERMICROPARTICLE W/W NOMINAL FORMULATION AQUEOUS POLYMER LOT (W/W ACTUAL)W/W EMULSION) PHASE M-22 2% (1.73%*, 2% MANNITOL Exendin-4 (50 mg/mL 4A50:50 1.74%^(#)) 0.5% in 80 mM AMMONIUM acetate buffer, SULFATE pH =4.0) 2% mannitol. 0.5% ammonium sulfate M-23 2% (1.66%*, 2% SUCROSEExendin-4 (50 mg/mL 4A 50:50 1.68%^(#)) 0.5% in 80 mM AMMONIUM acetateSULFATE buffer, pH = 4.0) 2% sucrose, 0.5% ammonium sulfate M-24 2%(1.86%^(#), 2% Exendin-4 (50 mg/mL 4A 50:50 1.90%*) TREHALOSE in 80 mM0.5% acetate AMMONIUM buffer, pH = 4.0) SULFATE 2% trehalose, 0.5%ammonium sulfate

[0201] The sustained release profiles in FIG. 12 show that mannitol andtrehalose tend to provide a higher Cmax than sucrose and higher plasmalevels over about the first week of release.

[0202] ACETATE BUFFER: The majority of formulations prepared included 80mM sodium acetate buffer in the inner aqueous phase duringmicroencapsulation. In this further formulation development, lowerlevels (30 mM) and also the absence of acetate buffer were evaluated.The release profile for Microparticle Formulations M-25-M-27 is depictedgraphically in FIG. 13. Inspection of the sustained release profile ofFIG. 13 suggest that the buffer can be removed from the formulationpossibly resulting in a reduced Cmax and without impacting the drugintegrity. The Microparticle Formulations M-25, M-26 and M-27 are setforth in Table 7. TABLE 7 % EXENDIN-4 IN % EXCIPIENT MicroparticleFORMULATION IN DRUG PHASE Formulation W/W NOMINAL FORMULATION (INNERAQUEOUS POLYMER Identifier (W/W ACTUAL) W/W EMULSION) PHASE M-25 2%(1.72%*, 2% Exendin-4 (50 mg/mL 4A 50:50 1.75%^(#)) MANNITOL in 80 mM0.5% acetate buffer, AMMONIUM pH = 4.0) SULFATE 2% mannitol. 0.5%ammonium sulfate M-26 2% (1.65%*, 2% Exendin-4 (50 mg/mL 4A 50:501.68%^(#)) MANNITOL in 0.5% WATER) AMMONIUM 2% mannitol, 0.5% SULFATEammonium sulfate M-27 2% (1.71%*, 2% Exendin-4 (50 mg/mL 4A 50:501.77%^(#)) TREHALOSE in 30 mM 0.5% acetate buffer, AMMONIUM pH = 4.0)SULFATE 2% trehalose, 0.5% ammonium sulfate

[0203] DESIGN-OF-EXPERIMENTS (DOE) APPROACH: Based on the discovery workdescribed above, a design-of-experiments approach was employed to selectformulations. Briefly, buffer was removed from the formulations, sugarcontent was fixed at 2%. The variables which were examined were drugload (2 or 3%), sugar type (sucrose or mannitol) and level of ammoniumsulfate (0.3 or 0.5%). The three variable, two-level design resulted ina total of 8 formulations from which the Selected Formulations, SF-1 andSF-2 were identified. The studies are set forth below.

[0204] DOE STUDY 1: Formulations containing 0.5% ammonium sulfate wereevaluated. The sustained release profile for the formulations are setforth in FIG. 14. One of the batches (2% drug load with mannitol) wasprepared at the 100 g scale and a precipitate was observed in theprimary emulsion possibly resulting in the higher Cmax observed. Thebioavailability was in the range of 15-30% for these formulations. Adescription of the Microparticle Formulations M-28-M-31 is set forth inTable 8. TABLE 8 % EXENDIN-4 DRUG PHASE IN % EXCIPIENT IN (INNERMICROPARTICLE FORMULATION FORMULATION AQUEOUS POLYMER LOT W/W ACTUAL W/WEMULSION) PHASE M-28 2.8% 2% SUCROSE Exendin-4 (50 mg/mL 4A 50:50 0.5%AMMONIUM in water) SULFATE 2% sucrose, 0.5% ammonium sulfate M-29 2.8%2% MANNITOL Exendin-4 (50 mg/mL 4A 50:50 0.5% AMMONIUM in SULFATE Water)2% mannitol, 0.5% ammonium sulfate M-30 1.8% 2% SUCROSE Exendin-4 (50mg/mL 4A 50:50 0.5% AMMONIUM in water) SULFATE 2% sucrose, 0.5% ammoniumsulfate M-31 1.9% 2% MANNITOL Exendin-4 (50 mg/mL 4A 50:50 0.5% AMMONIUMin water) SULFATE 2% mannitol, 0.5% ammonium sulfate

[0205] The Microparticle Formulations described in Table 8 were prepareda second time (Formulation M-28A, M-29A, M-30A and M-31A) but using adifferent drying method and evaluation of the serum profile determined.The sustained release profiles for M-28A-M-3 1A are depicted graphicallyin FIG. 15.

[0206] DOE STUDY 2: Formulations containing 0.3% ammonium sulfate wereevaluated. The sustained release profile for the formulations are setforth in FIG. 16. A 5 description of the Microparticle FormulationsM-32-M-35 is set forth in Table 9. TABLE 9 DRUG PHASE % EXENDIN-4 %EXCIPIENT IN (INNER MICROPARTICLE IN FORMULATION FORMULATION AQUEOUSPOLYMER LOT W/W ACTUAL W/W EMULSION) PHASE M-32  2.5% 2% SUCROSEExendin-4 4A 50:50 0.3% AMMONIUM (50 mg/mL in SULFATE water) 2% sucrose,0.3% ammonium sulfate M-33 2.66% 2% MANNITOL Exendin-4 (50 mg/mL 4A50:50 0.3% AMMONIUM in SULFATE Water) 2% mannitol, 0.3% ammonium sulfateM-34 1.66% 2% MANNITOL Exendin-4 (50 mg/mL 4A 50:50 0.3% AMMONIUM inSULFATE water) 2% mannitol, 0.3% ammonium sulfate M-35 1.68% 2% SUCROSEExendin-4 (50 mg/mL 4A 50:50 0.3% AMMONIUM in SULFATE water) 2% sucrose,0.3% ammonium sulfate

EXAMPLE 6 Effects of Local Delivery of Secondary Agent-ContainingMicroparticles on the Release of Exendin-4 from Exendin-ContainingMicroparticles

[0207] The effects on the pharmacokinetic profile of exendin-4 releasefollowing administration of exendin-4-containing microparticlesco-administered to male Sprague-Dawley rats with placebo microparticles,or triamcinolone-containing microparticles was determined.

[0208] Preparation of Exendin-Containing Microparticles andTriamcinolone-Containing Microparticles

[0209] Exendin-containing microparticles were prepared as describedabove for Selected Formulation 2 (SF-2) having 3% exendin-4 (in water),2% sucrose and 0.5% ammonium sulfate in 4A, 50:50 PLG and InitialFormulation-1 (IF-1) having 1% exendin-4 load (50 mg/mL Exendin-4), 1%sucrose (50 mg/mL sucrose) 30 mM sodium acetate (pH 4-4.5) and 3A, 50:50PLG.

[0210] Triamcinolone acetonide-containing microparticles (2% load) wereprepared as follows: 42 mg of triamcinolone acetonide was dissolved inbenzyl alcohol. The triamcinolone solution was then added to about 24.3mL of a 6% PLG (2.5A) solution in methylene chloride. The resultinghomogenous solution was added to a stirring solution of 5% PVA. Thestirring rate was raised until microscopic examination of the emulsionindicated that the diameter of the droplets was about 150-75 microns.The emulsion was then slowly added to stirring cold water. After about45 minutes of stirring, the suspension was allowed to settle at 4° C.The microparticles were collected by filtration, washed with cold water,frozen and lyophilized to dryness.

[0211] Placebo microparticles were prepared according to the process forpreparation of the triamcinolone microparticles but absent thetriamcinolone.

[0212] Administration of Microparticles

[0213] Microparticle administration was as follows: Animals wereanesthetized fully with 5% halothane. Each animal was shaved and theback swabbed with alcohol. Exendin-4 containing microparticles andeither placebo or triamcinolone-containing microparticles were suspendedin vehicle and injected into the interscapular site. Treatment groupsare summarized in Table 10.

[0214] A dose of 120 mg of exendin-4-containing microparticles(Formulation IF-1) plus 30 mg of placebo (Group A) or 10 mg of 2% w/wtriamcinolone-containing microparticles (Group B) was administered tothe rats. A dose of 40 mg of exendin-containing microparticles(Formulation SF-2) plus 30 mg of placebo (Group C) or 10 mg of 2% w/wtriamincinolone-containing microparticles (Group D) was alsoadministered to the rats. Sample collection time points were pre-bleed,2 hrs, 6 hrs, 10 hrs, and days 1, 2, 4, 7, 10, 14, 17, 21, 24, 29, 32,36 and 39. TABLE 10 EXENDIN-4 SECONDARY GROUP # ANIMALS MICROPARTICLESAGENT TREATMENT A 4 120 mg PLACEBO 30 mg IF-1 MICROPARTICLES B 4 120 mg2% TRIAMCINOLONE 10 mg IF-1 MICROPARTICLES C 4  40 mg PLACEBO 30 mg SF-2MICROPARTICLES D 4  40 mg 2% TRIAMCINOLONE 10 mg SF-2 MICROPARTICLES

[0215] Plasma Evaluation

[0216] To evaluate plasma exendin levels, 0.25 mL samples of plasma werecollected via tail vein on days 0 and 1, and 0.4 mL samples werecollected on the remaining days specified in Table 10 (four animals pergroup). The samples were centrifuged and the plasma fraction frozen(−80° C.). Plasma exendin levels were quantitated by IRMA.

[0217] Results

[0218] Plasma Exendin-4 Levels

[0219]FIG. 17 shows the results of the effects of exendin-4-containingmicroparticles co-administered with placebo microparticles andtriamcinolone acetonide-containing microparticles on plasma exendinlevels in the form of a graph of exendin plasma levels (pg/mL) versustime (days) post injection. As shown in FIG. 17, the pharmacokineticprofile for Group B was improved over controls (Group A). Specifically,enhanced bioavailability was observed for the triamcinolone acetonidetreated group (Group B) in that plasma levels on day 32 remaineddetectable while this was the last day detectable for the control group.It is noted that plasma levels were still detectable at day 39 for GroupB, showing a substantial increase in the duration of release of exendinwhen coadministered with triamcinolone acetonide-containingmicroparticles. C_(ave) levels, C_(max) and AUC were also desirablymodulated as a result of coadministration of triamcinoloneacetonide-containing microparticles with the exendin-containingmicroparticles.

[0220]FIG. 18 shows the results of the effects of exendin-containingmicroparticles co-administered with placebo microparticles andtriamcinolone acetonide-containing microparticles on serum exendinlevels in the form of a graph of exendin serum levels (pg/mL) versustime (days) post injection. As shown in FIG. 18, the pharmacokineticprofile for Group D was improved over controls, Group C. Specifically,enhanced bioavailability was observed for the triamcinolone treatedgroup (Group D) in that plasma levels were still detectable at day 39showing a substantial increase in the duration of release of exendinwhen coadministered with triamcinolone acetonide-containingmicroparticles in comparison to controls (Group C) which were notdetectable after day 24. C_(ave) levels, C_(max) and AUC were alsodesirably modulated as a result of coadministration of triamcinoloneacetonide-containing microparticles with the exendin-containingmicroparticles.

[0221] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A composition for the sustained release ofbiologically active polypeptide comprising: a biocompatible polymerhaving dispersed therein a biologically active polypeptide, a sugar anda salting-out salt.
 2. The sustained release composition of claim 1,wherein the polypeptide is selected from glucagon, glucagon-likepeptides, exendins, agonists of glucagon like peptides, vasoactiveintestinal peptide, immunoglobulins, antibodies, cytokines,interleukins, macrophage activating factors, interferons,erythropoietin, tumor necrosis factor, colony stimulating factors,insulin, enzymes, tumor suppressors, blood proteins, folliclestimulating hormone, growth hormone, adrenocorticotropic hormone, andluteinizing hormone releasing hormone, NGF, EGF, gastrin, GRH, defensin,enkephalins, and muteins, analogs, deletion and substitution variantsand pharmaceutically acceptable salts thereof.
 3. The sustained releasecomposition of claim 1, wherein the biologically active polypeptide is aglucoregulatory peptide.
 4. The sustained release composition of claim3, wherein the glucoregulatory peptide is selected from GLP-1, GLP-2,exendin-3, exendin-4 or a combination thereof.
 5. The sustained releasecomposition of claim 1, wherein the biologically active polypeptide ispresent from about 0.01% (w/w) to about 50% (w/w) of the total weight ofthe composition.
 6. The sustained release composition of claim 5,wherein the biologically active polypeptide is present in a range fromabout 0.1% (w/w) to about 30% (w/w) of the total weight of thecomposition.
 7. The sustained release composition of claim 6, whereinthe polypeptide is present from about 0.1% (w/w) to about 10% (w/w) ofthe total weight of the sustained release composition.
 8. The sustainedrelease composition of claim 7, wherein the polypeptide is present fromabout 0.5% (w/w) to about 5% (w/w) of the total weight of the sustainedrelease composition
 9. The sustained release composition of claim 1,wherein the sugar is present from about 0.01% to about 50% w/w of thetotal weight of the sustained release composition.
 10. The sustainedrelease composition of claim 9, wherein the sugar is present from about0.01% to about 10% w/w of the total weight of the sustained releasecomposition.
 11. The sustained release composition of claim 10, whereinthe sugar ins present from about 0.1% to about 5% w/w of the totalweight of the sustained release composition.
 12. The sustained releasecomposition of claim 1, wherein the sugar is selected from amonosaccharide, a disaccharide, a sugar alcohol or a combinationthereof.
 13. The sustained release composition of claim 12, wherein thesugar is selected from sucrose, trehalose, mannitol and combinationsthereof.
 14. The sustained release composition of claim 12, wherein thesugar is a disaccharide.
 15. The sustained release composition of claim14, wherein the disaccharide is sucrose, trehalose or a combinationthereof.
 16. The sustained release composition of claim 1, wherein thesalting-out salt comprises a salt containing a cation selected fromMg⁺², Li⁺, Na⁺, K⁺ and NH₄ ⁺ and combinations thereof.
 17. The sustainedrelease composition of claim 1, wherein the salting-out salt comprises asalt containing an anion selected from SO₄ ⁻², HPO₄ ⁻², acetate,citrate, tartrate, Cl⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻ and SCN⁻ andcombinations thereof.
 18. The sustained release composition of claim 1,wherein the salting-out salt is ammonium sulfate.
 19. The sustainedrelease composition of claim 1, wherein the salting-out salt is presentfrom about 0.01% to about 50% w/w of the total weight of the sustainedrelease composition.
 20. The sustained release composition of claim 19,wherein the salting-out salt is present from about 0.01% to about 10%w/w of the total weight of the sustained release composition.
 21. Thesustained release composition of claim 1, wherein the biocompatiblepolymer is selected from the group consisting of poly(lactides),poly(glycolides), poly(lactide-co-glycolides), poly(lactic acid)s,poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s,polycaprolactone, polycarbonates, polyesteramides, polyanhydrides,poly(amino acids), polyorthoesters, polycyanoacrylates,poly(p-dioxanone), poly(alkylene oxalate)s, biodegradable polyurethanes,blends thereof and copolymers thereof.
 22. The sustained releasecomposition of claim 21, wherein said polymer comprisespoly(lactide-co-glycolide).
 23. The sustained release composition ofclaim 1, further comprising a corticosteroid.
 24. The sustained releasecomposition of claim 23, wherein the corticosteroid is selected from21-Acetoxypregnenolone, Alclometasone, Algestone, Amcinonide,Beclomethasone, Betamethasone, Budesonide, Chloroprednisone, Clobetasol,Clobetasone, Clocortolone, Cloprednol, Corticosterone, Cortisone,Cortivazol, Deflazacort, Desonide, Desoximetasone, Dexamethasone,Disflorasone, Diflucortolone, Difluprednate, Enoxolone, Fluazacort,Flucloronide, Flumethasone, Flunisolide, Flucinolone Acetonide,Fluocinonide, Fluocortin Butyl, Flucortolone, Fluorometholone,Fluperolone Acetate, Fluprednidene Acetate, Fluprednisolone,Flurandrenolide, Fluticasone Propionate, Formocortal, Halcinonide,Halobetasol Propionate, Halometasone, Halopredone Acetate,Hydrocortamate, Hydrocortisone, Loteprednol Etabonate, Mazipredone,Medrysone, Meprednisone, Methylprednisolone, Mometasone Furoate,Paramethasone, Prednicarbate, Prednisolone, Prednisolone25-Diethylamino-acetate, Prednisolone Sodium Phosphate, Prednisone,Prednival, Prednylidene, Rimexolone, Tixocortol, Triamcinolone,Triamcinolone Acetonide, Triamcinolone Acetonide 21-oic acid methylester, Triamcinolone Benetonide, Triamcinolone Hexacetonide,Triamcinolone Diacetate, pharmaceutically acceptable mixtures and saltsthereof.
 25. The sustained release composition of claim 24, wherein thecorticosteroid is selected from Triamcinolone, Triamcinolone Acetonide,Triamcinolone Acetonide 21-oic acid methyl ester, TriamcinoloneBenetonide, Triamcinolone Hexacetonide, Triamcinolone Diacetate,pharmaceutically acceptable mixtures and salts thereof.
 26. Thesustained release composition of claim 23, wherein the corticosteroid isco-incorporated into the sustained release composition.
 27. Thesustained release composition of claim 23, wherein the corticosteroid isseparately incorporated into a second biocompatible polymer.
 28. Thesustained release composition of claim 27, wherein the secondbiocompatible polymer is the same as the biocompatible polymer of thesustained release composition.
 29. The sustained release composition ofclaim 27, wherein the second biocompatible polymer is different from thebiocompatible polymer of the sustained release composition.
 30. Thesustained release composition of claim 23, wherein the corticosteroid isunencapusulated but commingled with the sustained release composition.31. A composition for the sustained release of biologically activepolypeptide comprising: a biocompatible polymer having dispersed thereinexendin-4, sucrose and ammonium sulfate.
 32. The composition of claim31, wherein the biocompatible polymer is selected from poly(lactides),poly(glycolides), poly(lactide-co-glycolides), poly(lactic acid)s,poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s and blends andcopolymers thereof.
 33. The composition of claim 31, wherein the sucroseis present at a concentration from about 0.01% w/w to about 10% w/w ofthe total weight of the sustained release composition.
 34. Thecomposition of claim 31, wherein the ammonium sulfate is present at aconcentration of from about 0.01% w/w to about 10% w/w of the totalweight of the sustained release composition.
 35. The sustained releasecomposition of claim 31, wherein the exendin-4 is present at aconcentration of about 0.1% to about 10% of the total weight of thecomposition.
 36. A method of treating a patient suffering from Type 2diabetes comprising administering a therapeutically effective amount ofa sustained release composition comprising a biocompatible polymerhaving dispersed therein a biologically active exendin-4, a sugar and asalting-out salt.
 37. The method of claim 36, wherein the biocompatiblepolymer of the sustained release composition is selected frompoly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s and blends and copolymers thereof.
 38. The method of claim 36,wherein the sugar is present in the sustained release composition at aconcentration from about 0.01% w/w to about 10% w/w of the total weightof the sustained release composition.
 39. The method of claim 36,wherein the salting-out salt in the sustained release composition ispresent at a concentration of from about 0.01% w/w to about 10% w/w ofthe total weight of the sustained release composition.
 40. The method ofclaim 36, wherein the exendin-4 is present in the sustained releasecomposition at a concentration of about 0.1% to about 10% of the totalweight of the composition.